Pyridostigmine is a synthetic quaternary ammonium compound which occurs as a hygroscopic crystalline powder, freely soluble in water and alcohol. Being a carbamate, pyridostigmine has a characteristic odor, a bitter taste and is unstable in alkaline solutions. Aqueous solutions of pyridostigmine may be sterilizes by autoclaving. Pyridostigmine has been found to have a profound effect on the neurological functions of humans and other mammals.
Pyridostigmine is a reversible cholinesterase inhibitor which prevents the hydrolysis of acetylcholine by competing with acetylcholine for attachment to acetylcholinesterase. Acetylcholine is a neurotransmitter stored in vesicles where it isprimarily released by nerve impulses. The vesicles migrate towards the terminal synaptic membrane during nerve stimulation and disgorge acetylcholine by exocytosis Upon release fromthe cholinergic nerve endings, acetylcholine is inactivated by enzymatic degradation. The inactivation is accomplished by the hydrolysis of acetylcholine by cholinesterase. The specific cholinesterase for acetylcholine, acetylcholinesterase, is quite efficient--one molecule of the enzyme is able to hydrolyze 3.times.105 molecules of acetylcholine per minute.
Because a pyridostigmine-acetylcholinseterase enzyme complex hydrolyzes at a much slower rate than the corresponding acetylcholine-acetylcholinesterese enzyme complex, acetylcholine accumulates at the cholinergic synapses. Due to the reversible nature, i.e., uncoupling, of the pyridostigmine-acetylcholinestebase complex, pyridostigmine appears to facilitate the transmission of impulses across the myoneural junction In other words, the introduction of pyridostigmine produces generalized cholinergic responses. And while pyridostigmine has a direct cholinomimetic effect on skeletal muscles, because of its quaternary ammonium structure, moderate doses of pyridostigming usually do not effect the central nervous system.
Reversible cholinesterase inhibitors, such as pyridostigmine, have been proposed as antidotes to nerve agents used in chemical warfare. Many nerve agents, including sarin, soman, tabun and VX, are organ-phosphorous compounds which, while liquid at room temperature, are readily vaporized under normal atmospheric conditions. The extreme toxicity of such organo-phosphorous compounds are related to their short-lived, but irreversible destruction of the functioning of nerves and organs. By phosphorylating acetylcholinesterase, organophosphorous compounds such as varin form stable, irreversible Complexes with acetylcholinesterase. The formation of such stable complexes permanently prevent the normal function of acetylcholinesterase, i.e., the termination of acetylcholine actions at synaptic, particularly neuromuscular, junctions. Since the enzyme is completely and permanently prevented from binding with acetylcholine, the acetylcholine quickly accumulates at receptor sites to a degree sufficient to produce loss of function in target nervos and organs.
From animal studies, it has been proposed that nerve agent toxicity can be prevented by the preadministration of a short acting, reversible cholinosterase inhibitor such as pyridostigmine. The pyridostigmine would temporarily bind acetylcholinesterase in the tissue which would prevent its phosphorylation by the nerve agents and the resulting irreversible inactivation of the active site of the acetylcholinesterase.
Moreover, there are several clinical uses for reversible cholinesterase inhibiting agents such as pyridostigmine. Pyridostigmine is used to improve muscle strength in the symptomatic treatment of mysthenia gravis. Parental pyridostigmine is also useful in reversing of the effects of nondepolarizing neuromuscular blocking agents, e.g., tubocurarine, metocurine, gallamine or pancuronium, after surgery. Recently, cholinesterase inhibitors have been used in an attempt to reverse certain regenerative disorders of the central nervous system. It has been suggested that since cOgnitive changes observed during the aging process, e.g., Alzheimer's syndrome, may be related to gradual reductions in acetylcholine in various parts of the brain, the administration of pyridostigmine might help reduce or reverse the observed cognitive changes.
Results obtained from the administration of oholinesterase inhibitors such as pyridostigmine to maintain, restore or increase acetylcholine levels in patients, including those with Alzheimer's syndrome, have been equivocal. A primary problem encountered with the clinical use of pyridostigmine has been that it is toxic at levels very close to those which produce therapeutic results. For example, in certain patients pyridostigmine has been associated with adverse effects typical of exaggerated responses to parasympathetic stimulation including adverse muscarinic effects such as nausea, vomitin , diarrhea, miosis, excessive salivation and sweating, abdomimal cramps, bradycardia, bronchial secretion and bronchospasm. Other side effects of pyridostigmine include generalized weakness, muscle cramps, fasciculation, hypotension, and if administered intraveneously, thrombophlebitis. A substantial over administration of pyridostigmine causes cholinergic crisis leading to death.
Despite its relatively long clinical use, relatively little is known about the pharamacokinetic paramenters, i.e., the change in concentration at various sites, including absorption, distribution, metabolism and excretion, of pyridostigmine in man. The death of information is due primarily to the lack of a satisfactory analytical method for measuring low concentrations of pyridostigmine in biological fluids.
There are several reasons why analytical methods for measuring pyridostigmine in biological fluids have been unsatisfactory. The amounts of pyridostigmine to be measured are extremely low, which means that any analytical method must be sufficiently sensitive to detect extremely low levels of pyridostigmine. Moreover, the extensive in vitro hydrolysis of pyridostigmine which may take place in biological solutions, particularly in plasma and blood, exacerbates the problems associated with measuring small quantities of pyridostigmine. Consequently, analytical techniques such as paper chromatography, spectrophotomety, gas chromatography and high pressure liquid chromatography have proven to be unsatisfactory.
While immunoassays are often used to determine the minute quantities of drugs in biological fluids, heretofore an immunoassay for determining thepresence of pyridostigmine in such fluids has been unavailable. The pyridostigmine molecule is too small to stimulate the immune systems of animals normally used to produce antibodies. The problem is complicated by the fact that the pyridostigmine molecule does not contain any functional groups which are usually necessary for a molecule to be linked to a protein immunogen. Of course, to be effective, an immunogen for pyridostigmine has to be sufficiently selective to avoid cross reactivity with the metabolites of pyridostigmine.
It is therefore an object of the present invention to provide for a method for the determination of the amount of pyridostigmine in biological fluid.
It is another object of the present invention to provide an immunoassay for the determination of the amount of pyridostigmine in biological fluids.
Yet another object of the present invention is to provide an immunogen which after injection into animals will result in the production of antibodies for use in an immunoassay for the determination of pyridostigmine in biological fluids.
Still a further object of the present invention is to provide monoclonal and polyclonal antibodies which can be used to determine pyridostigmine in biological fluids.
A further object of the present invention is to provide for a method of measuring pyridostigmine in biological fluids which faoilitates the study of the pharmacokinetics of pyridostigmine.
Still another object of the present invention is to proVide for a reduction in the toxicity and other adverse side effects which results from the clinical use of pyridostigmine.
These and other objects of the present invention, as will become more readily apparent hereinafter, are achieved by the invention described herein below.